Mitosis is one of the most fundamental processes of eukaryotic cells. In mitosis duplicate copies of the genetic material are separated into the new daughter cells. Obviously, mitosis is of central importance for the development, even the basic existence of eukaryotic organisms. Abnormal mitosis can be lethal or can result in abnormal development or malignancy. We do not know how mitosis is regulated. Microtubules (MTs) are the major structural component of the mitotic apparatus (MA). Controlled disassembly of MTs in the MA is important for the normal process of mitosis. Calcium ion (Ca++) causes disassembly of MTs and evidence suggests that calmodulin (CaM) may mediate the effect of Ca++ on MTs. CaM is found in the MA and Ca++ is concentrated in membrane-bound vesicles in the MA. Therefore, it has been hypothesized that CaM and Ca++ may regulate MT structure and function during mitosis. Preliminary experiments indicate, however, that CaM may not be associated with MTs of the MA, thus suggesting that CaM may not be acting directly on MTs as is generally believed. In an effort to learn about the role played by CaM in the MA, it is proposed that studies be conducted to examine the location and proximity within the MA of CaM, Ca++ and MTs and to study the effects on mitosis of CaM inhibitors and of alteration of Ca++ in the MA. To accomplish these aims, CaM and MTs will be observed in live cells by microinjection of fluorescently labeled CaM and tubulin. The distribution of the proteins will be observed and studied using low light video recording and image analysis methods. CaM will also be localized at the EM level by a new method. Ca++ will be detected and quantitated using the calcium-specific fluorescent probe quin2. Proximity of CaM and MTs will be detected by fluorescence energy transfer between pairs of appropriately labeled fluorescent protein derivatives. To examine the effects of CaM inhibitors and competitors, cells will be microinjected with CaM inhibitors such as R24571 or trifluoperazine or with CaM-binding proteins such as CaM antibody, Fab antibody fragments, troponin I or melittin to block CaM action in the MA. Effects on mitosis will be detected by time-lapse video recording of injected cells, with rates of chromosome separation in anaphase being quantitated and used as a measure of MA function. It is expected that the results of these studies will contribute to knowledge of the role played by CaM and Ca++ in the control of mitosis and regulation of the MA. Such knowledge may open new avenues of approach to the development of rational therapies for control of malignancies.